Blood-pressure-lowering substance and process of producing it



' RALPH H. MAJOR, OF KANSAS CITY, KANSAS, ASSIGNOR TO OF INDIANAPOLIS,INDIANA, A CORPORATION OF Patented Aug. 1932 umrsn sures P TENTfo-Fiucst ELI LILLY m courm,

manna BLOOD-PBESSUBE-LOWERINQ SUBSTANCE AND PROCESS OI PRODUCING IT NoDrawing Application and April 10,

It is the object of my invention to obtain from the liver an effectiveproduct containing ina. high degree of concentration, and in sufiicientpurity so that it may be repeatedly administered to human beings withoutdangerous shock or undue toxic eflects, the active principle or hormonewhich reduces high blood pressure (hypertension) and resymptoms.

lieves certain subjectivesymptoms commonly associated with hypertension,such as headache, insomnia, nocturia, tinnitus, etc.

Previous investigators have suggested that the liver contains orsecretes a substance or hormone which has a depressor etfect on bloodpressure and/or relieves such subjective Indeed, previous investigatorshave produced extracts from the liver that exerted some depressoraction. The followmg references-are some of those that describe thisprevious work:

1. La Presse Medicale, Mercredi 24 Mai 1922, by H. Roger;

2, The American Journal of Physiology,

June 1, 1915, by Fawcett, Rogers,Rah'e and Beebe;

3 The Journal of Pharmacolo andExperlmental Therapeutics, Vol. II 0. 6,July, 1911, page 507, by Voegtlin and Bernheim;

4. The Journal of Pharmacolo and Exo. 4, July, 1919, gage 301, byVoegtlin and ll: ers;

5. outhern Medical Journal, March 1922, and'Vol, XVI, November, 1923, byA. I. Levin,

All such previous extracts, however, so far as I am aware, havebeenineffective or unsafe for continuous therapeutic use in reallyalleviatin hi h blood pressure, or for relieving such sub ectlvesymptoms commonly associated with hypertension, such asheadache,'insomnia nocturia, tinnitus, etc.; probably either becausethey have contained too little of the non-toxic blood-pressure-loweringsub stance to beef any real benefit, or because they-have had such ahigh proportion of peptone, choline, histamine-like substances, proteinsplit products, etc. (which are toxic blood-pressure-lowerin substances,that upon administration t ey have pro uced a violent and immediatetoxic or shock effect,

1928. Serial No. 101,191.

which made them dan rous to use at all and impossible to use in su cientquantit to produce the non-toxic blood-pressureowering effect or torelieve such sub ective symptoms, even in single doses,not to mentionthe cumulative difliculties from repeated doses.

This situation has been complicated b the fact that the shock or toxicefiectmamfests itself in a sudden but temporary lowering of the bloodpressurethat is, temporary in cases where this sudden eifect is notfatal. This shock efi'ect usually takes place within a few seconds afteradmlnistration of the product, and if the amount of such im urities issufficientit may sharply lower t e. blood pressure to a point wheredeath occurs. This sharp lowering of the blood pressure in shock may belessened by giving a smaller dose, so that such lowerin will not reducethe blood pressure to the atal point; in which .case the blood ressurerecovers from the shock' within a ew minutes, and returns tosubstantially what it was before the dose was given, or even higher. Itis only subse uent to that that the-'non-toxigefiect of the boodpr'essure-lowering hormone or active-principle is obtained,-as sucheffect starts slowly v, and continues over a period of from severaltors, so far as I am aware, have not been a le to obtain from the livera substance which could be administered safely and repeatedly to producta- 'prolongedtherapeutic effect of value in the treatment ofhypertension;

because they either did not obtain potent extracts, or did not obtainextracts su-fiiciently free from toxic impurities to make it pos-.

sible to administer doses of sufficient size to give such desiredprolonged therapeutlc efects.

In contradistinction to this, I have ob tained from the liver an extractwhich is quite efi'ective therapeutically in reducing high bloodpressure and/or relieving certain su jective symptoms commonlyassociated with hypertension, such as headache, insomnia, nocturia,tinnitus, etc., over a fairly prolonged time, and one which can be used,and used repeatedly, without producing ill effects in the patient, andespecially without producing any dangerous amount of shock. That is, theliver. extract which I have roduced is not only potent to reduce hi hBlood pressure and/or to relieve such su jective symptoms, but can begiven in doses of sufiicient size to be therapeutically effective,because it .is sufiiciently free from proteins and other impurities 'sothat any 7 sudden shock-produced lowering in the blood pressure isrelatively small, is not sufficient to reduce blood pressure to a pointdangerously lower than normal blood pressure, and is ordinarily notsuflicient to make the sudden shock-produced lowering greater than thelater and longer continued lowering which is the therapeutic effect ofthe hormone.

In carrying out my invention, I may use liver from any availableanimals, as from hogs or beef. I mince or grind a quantity of suchliver, either liver from freshly slaughtered animals or liver which hasbeenimmediately refrigerated alter removal from freshly slaughteredanimals and kept refrigerated until used. Then I extract this liver witha solvent which takes up from the liver the.

desired blood-pressure-lowering hormone or active principle, or somesubstance containing it, and which, in addition, in some wa eitherinhibits the deleterious action on sucii hormone or active principle ofany copresent destructive agents (such as protcolytic enzymes) ordestroys such agents. The extract thus obtained, after being separatedfrom the residual solid matter, is treated to remove the major portionof any undesirable contents. Whilethis treatment includes other steps,probably the most important part' of the procedure is the separation ofthe undesirable matter, mostly protein matter, by fractionation withvarying concentrations of alcohol. The matter which is removed atalcoholic concentrations up to about is discarded; but this 70% filtrateor the precipitate-fractions obtained at higher alcohol concentrations,or both this filtrate and these precipitate-fractions, are preserved, asthey contain at least a large part of the blood-pressure-loweringhormone or active principle. While this fractional precipitation withalcohol .has removed a large part of the undesirable matter, furthertreatment may also be given the preserved substances, for furtherpurification.

In order that a better understanding of my invention may be obtained, Ishall now equal volume of 95% alcohol; by which the alcoholconcentration is raised. A precipitate, which maybe called precipitateA, forms as a result of this increased alcohol concentration. v

This precipitate A is suitably removed, as by filtration. To theresultant filtrate, which may be called filtrate No. 2, is added anequal volume of 95% alcohol, which raises the alcohol concentrationstill higher.

Another precipitate forms as the result of this increased alcoholconcentration; and this precipitate may be called precipitate B.

This precipitate B is likewise suitably removed, as by filtration; butis preserved, as it contains a large partof the desiredbloodpressure-lowering hormone. The filtrate from this lastprecipitation, which filtrate may be called filtrate o. 3, is alsopreserved; as it also contains some of the desired hormone. Bothprecipitate B and filtrate No. 3 are desirably used as a basis for finalextracts, after additional purification if desired.

Thus the precipitate B may be dissolved in distilled water, usually of avolume of about one-twentieth of the volume of the original extractingliquid. This solution may be appropriately treated with various solventswhich do not mix with water, in order that a still higher purificationmay be obtained. For instance, it may be so treated, separately, withether, chloroform, amyl alcohol,-lead acetate, and hydrogen sulphide.Any of these treatments and separations may be repeated several times,if desired; and, if desired, the treatments may be interspersed, as byhaving a treatment with ether first, then a treatment with chloroform,then several treatments with amyl alcohol, and then another treatmentwith ether. The aqueous solution thus obtained, which contains thehormone or active principle, is filtered to remove any precipitate orcloudiness, and the resultant filtrate used as the purified extract.

Likewise, the filtrate No. 3 may also be subjected to furtherpurification. To this end, such filtrate is desirably evaporated todryness in a water bath; and this residue treated All the purifiedextracts which have been thus obtained have been demonstrated to have adepressor action on the blood pressure in animals, and in man so far asthey have been used in man. They haVe been used to 5 depresshypertension in man; and have been used in animals to counteract theeflects of artificial hypertension, as producedboth by pituitaryextracts and by guanidine compounds. Theyl not only-reduce both naturaland artificial ypertension, but they can be given in sufficient doses todo this markedly without producing undue shock effect; andso areindicated in the treatment ofhypertension. a v

I claim as my invention Y 1. The method of producing ablood-pressure-lowering substance from liver, which consists inextracting the liver with alcohol, and separating theblood-pressure-lowering substance from much of the contaminating matterextracted with it by fractionation with varying concentrations ofalcohol.

'2. The method of producing a-blood-pres sure-lowering substance fromliver, which consists in extractin the liver with alcohol,separating'the bloo -pressure-lowering subhaving the property ofreducing the subjective symptoms commonly associated with hypertension,such as headache, insomnia,

nocturia, tinnitus, etc., and sufliciently free 1 RALPH MAJOR.

stance from much of the contaminating matter extracted with' it byfractionation with varying con'centrations'of alcohol, and form-' inga'solution from a precipitate obtained at higher alcohol concentration.

3. The method of producing a blood-pressure-lowering substance fromliver, which consists inextractin the liver with alcohol, separating thebloo -pressure-lowering substance from much of the contaminatingmathundred and ter extracted with it by fractionation with varyingconcentrations of alcohol, forming solutions from precipitates obtainedat higher alcohol concentration, and using both the solution and thefiltrate remaining after a precipitation at a higher alcohol concentra-3 tion as a basis for them eutic extracts.- Y 4. A substance derive fromthe liver and 45 having a prolonged blood-pressure-lowering effect wheninjected'in cases of essential hypertension and a sufiiciently smallshort actin blood-pressure-lowering efiect to en able oses ofsuflicientsize to (L-duce such prolonged efiect. 5. A substance deri.

suificiently free from may without substantial seam-the livei-and havinga blood-pressure lowering"'efiect, and

be giventoproimlpuritieaso that it v ali'gerbeadm" -55 tered repeatedlyin sufiicientquantities to counteract excessive blood 'ressure. 6.Asubstance derived.

, having a blood-pressure -lowering efi'ect, and

sufliciently free from im 'peutically efiective doses. to a patient withhigh blood pressure will; nqt cause a sudden reduction in blood pressureto the danger the liver and fie -s0 that the 0 shook efiectfromadmimstering it "in there'-

